Nikon scope pictures annotation & data collection

Annotation checklist

This guide is meant to be a checklist for annotating categories of embryonic development in Montipora capitata using NIS-Elements BR.

For a basic introduction to using NIS Elements Annotation & Measurement tool, check out this elearning video.

1. Open NIS-Elements BR

2. File>Open…

3. Select sample folder

4. Open *stitch*.nd2

5. Show scale> Burn scale…

   1. Select No in response to pop-up asking to convert to RGB

6. Select Appending Labels and click ont he three dots to change font color to white and background to transparent. Keep all other settings the same.

   

7. Using the annotation & measurement tool, insert text to label via numbering 1-n each embryo at its upper left quadrant. Do not number debris.

8. Click on the taxonomy shape icon under Count & Taxonomy

9. Right click inside the table to access Taxonomy Options

10. Set number of classes to 10

    1. egg

    2. cleavage

    3. 4 to 16 cell

    4. 32 to 64 cell

    5. prawn chip

    6. early gastrula

    7. debris

    8. malformed

    9. typical

    10. torn

        

11. Click on taxonomy label and label each embryo with a selection from phase (egg, cleavage, 4-16, 32-64, prawn chip, or early gastrula) and proper selection from status (debris, malformed, typical, torn)

12. File > Save

    1. This saves the annotation layer to the .nd2 file

13. File > Save As … > JPEG - JIFF Format (*.jpg; *.jpeg)

    1. Filename _scale replaces _00

       

14. Close jpg, make sure you’re working on the nd2 file with annotations showing

15. Save again as JPG with Burn Annotations checked, and anno to replace scale

16. Reopen .nd2 file with annotations showing

17. Fill out google sheet

18. Save Google Sheet

19. egg (unfertilized oocyte)

20. cleavage (3-4 hours post fertilization)

21. 4-16 cell stage

22. 32-64 cell stage

23. prawn chip (9 hours post fertilization)

24. early gastrula (14 hours post fertilization)

Note that the phases in bold are the ones that we expect our embryos to be in based on hours post fertilization that they were preserved in 4% ZFIX. In this study, we conducted bundle-bundle crosses in 20mL of treatment solution (either a 0.22 micron filtered seawater control, or three increasing levels of PVC leachate derived from microplastics: 0.01 mg/L PVC leachate low, 0.1mg/L PVC leachate mid, or 1mg/L PVC leachate high).

Oocyte

exemplar

fragmented

deformed

Cleavage

exemplar

fragmented

deformed

4-16 cell

exemplar

fragmented

deformed

32-64 cell

exemplar

fragmented

deformed

Prawn chip

exemplar

fragmented

deformed

Early gastrula

exemplar

fragmented

deformed

Gastrula

exemplar

fragmented

deformed